Biology subjects

Palermo, J.

Publications and source records attributed to Palermo, J..

6 recordsLinked to original sources

bioRxiv2025

Cross-species variant-to-function analyses implicate MEIS1 in conferring sleep abnormalities and impaired cerebellar development

Genome wide association study (GWAS) reports substantially outpace subsequent functional characterization. Pinpointing the causal effector gene(s) at GWAS loci remains challenging given the non-coding genomic residency of >98% of these signals. We previously implicated effector genes at GWAS loci for the complex and polygenic disorder of human insomnia using a high-resolution cell-specific, chromatin capture-based variant-to-gene mapping protocol, paired with sleep phenotyping in Drosophila. In this study, we leveraged a diurnal vertebrate model with higher genomic conservation, namely zebrafish, to screen our six highest confidence candidate genes and identify those whose loss-of-function impaired sleep characteristics related to human insomnia-like behaviors. Of these genes, we observed that CRISPR-mediated deletion of the zebrafish ortholog of MEIS1 produced nighttime specific sleep fragmentation and increased latency to sleep, pointing to a conserved role for MEIS1 in sleep maintenance. Comparing our human cell-based chromatin accessibility and contact maps with publicly available zebrafish spatial genomic data revealed highly conserved genomic architecture harboring the insomnia GWAS variant of interest. Notably, this genomic conservation was selective for the zebrafish ortholog which contributed to the sleep phenotype, meis1b, while the duplicated ohnolog meis1a proved dispensable. Motivated by this, we characterized the spatio-temporal expression of meis1b in zebrafish, showing it is comparable to human with respect to cerebellar granule progenitors. Ultimately, we found that loss of meis1b impairs cerebellar development. Together, our work provides a powerful model for screening human disorder risk genes for sleep fragmentation using a tractable vertebrate and supports a conserved cerebellar role for MEIS1 in sleep disturbance.

neuroscience↗

bioRxiv2023

Perturbation of the insomnia WDR90 GWAS locus pinpoints rs3752495 as a causal variant influencing distal expression of neighboring gene, PIG-Q

Although genome wide association studies (GWAS) have been crucial for the identification of loci associated with sleep traits and disorders, the method itself does not directly uncover the underlying causal variants and corresponding effector genes. The overwhelming majority of such variants reside in non-coding regions and are therefore presumed to impact the activity of cis-regulatory elements, such as enhancers. Our previously reported variant-to-gene mapping effort in human induced pluripotent stem cell (iPSC)-derived neural progenitor cells (NPCs), combined with validation in both Drosophila and zebrafish, implicated PIG-Q as a functionally relevant gene at the insomnia WDR90 locus. However, importantly that effort did not characterize the corresponding underlying causal variant at this GWAS signal. Specifically, our genome-wide ATAC-seq and high-resolution promoter-focused Capture C datasets generated in this cell setting brought our attention to a shortlist of three tightly neighboring single nucleotide polymorphisms (SNPs) in strong linkage disequilibrium in a candidate intronic enhancer region of WDR90 that contacted the open PIG-Q promoter. The objective of this study was to investigate the influence of the proxy SNPs collectively and then individually on PIG-Q modulation and to pinpoint the causal "regulatory" variant among the three SNPs. Starting at a gross level perturbation, deletion of the entire region harboring all three SNPs in human iPSC-derived neural progenitor cells via CRISPR-Cas9 editing and subsequent RNA sequencing revealed expression changes in specific PIG-Q transcripts. Results from more refined individual luciferase reporter assays for each of the three SNPs in iPSCs revealed that the intronic region with the rs3752495 risk allele induced a [~]2.5-fold increase in luciferase expression (n=10). Importantly, rs3752495 also exhibited an allele specific effect, with the risk allele increasing the luciferase expression by [~]2-fold compared to the non-risk allele. In conclusion, our variant-to-function approach and subsequent in vitro validation implicates rs3752495 as a causal insomnia risk variant embedded at the WDR90-PIG-Q locus.

genomics↗

bioRxiv2022

Zebrafish screen of high-confidence targets at insomnia GWAS loci implicates MEIS1, SKIV2L, and ARFGAP2 as conserved regulators of sleep-wake behaviors

Recent large-scale human genome-wide association studies (GWAS) for insomnia have identified more than 200 significant loci. The functional relevance of these loci to the pathogenesis of insomnia is largely unknown. GWAS signals are typically non-coding variants, which are often arbitrarily annotated to the nearest protein-coding gene; however, due to 3D chromatin structure, variants can interact with more distal genes driving their function. The distal gene may, therefore, represent the true causal gene influencing the phenotype. By integrating our high-resolution chromatin interaction maps from neural progenitor cells with phenotypic data from a Drosophila RNAi screen, we prioritized candidate genes that we hypothesized would have deep phylogenetic conservation of sleep function. To determine the conservation of these candidate genes in the context of vertebrate sleep and their relevance to insomnia-like behaviors, we performed CRISPR-Cas9 mutagenesis in larval zebrafish for six highly conserved candidate genes and examined sleep-wake behaviors using automated video-tracking. CRISPR mutation of zebrafish orthologs of MEIS1 and SKIV2L produced insomnia-like behaviors, while mutation of ARFGAP2 impaired activity and development in our larval zebrafish model, demonstrating the importance of performing functional validation of GWAS-implicated effector genes to reveal genes influencing disease-relevant mechanisms.

genetics↗

bioRxiv2021

Variant-to-gene-mapping followed by cross-species genetic screening identifies GPI-anchor biosynthesis as novel regulator of sleep

Sleep is nearly ubiquitous throughout the animal kingdom, with deficiencies in sleep having been linked to a wide range of human disorders and diseases. While genome wide association studies (GWAS) in humans have identified loci robustly associated with several heritable diseases or traits, little is known about the functional roles of the underlying causal variants in regulating sleep duration or quality. We applied an ATAC-seq/promoter focused Capture C strategy in human iPSC-derived neural progenitors to carry out a variant-to-gene mapping campaign that identified 88 candidate sleep effector genes connected to relevant GWAS signals. To functionally validate the role of the implicated effector genes in sleep regulation, we performed a neuron-specific RNAi screen in the fruit fly, Drosophila melanogaster. This approach identified a number of genes that regulated sleep, including phosphatidylinositol N-acetylglucosaminyltransferase subunit Q (PIG-Q), a gene that encodes an enzyme involved in the first step of glycosylphosphatidylinositol (GPI)- anchor biosynthesis. We show that flies deficient for PIG-Q have longer sleep during both day and night due to an increase in the total number of sleep bouts. Subsequent systematic investigation of other PIG-family genes identified increased sleep in flies for multiple different genes within the PIG pathway. We then mutated the PIG-Q locus in zebrafish and identified similar increases in sleep to those observed in Drosophila, confirming deep homology of PIG-Q mediated sleep regulation. These results provide the first physical variant-to-gene mapping of human sleep genes followed by a model organism-based prioritization, revealing a novel and conserved role for GPI-anchor biosynthesis in sleep regulation.

genetics↗

bioRxiv2021

Using Human Induced Pluripotent Stem Cell Derived Organoids to Identify New Pathologies in Patients with PDX1 Mutations

Two patients with mutations in PDX1 presented with pancreatic agenesis, chronic diarrhea and poor weight gain, the causes of which were not identified through routine clinical testing. We generated patient derived organoids as a novel diagnostic strategy and observed that PDX1188delC/188delC antral organoids convert to an intestinal phenotype, while intestinal organoids undergo gastric metaplasia with significant reduction in enteroendocrine cells. This prompted a re-examination of gastric and intestinal biopsies from both PDX1188delC/188delC patients, which recapitulated organoid phenotypes. Antral biopsies had increased parietal cells and lacked G-cells suggesting loss of antral identity. These patients will now be monitored for the progression of metaplasia. This study demonstrates the utility of organoids for patient diagnoses and treatment.

developmental biology↗

bioRxiv2020

Ir56d-dependent fatty acid responses in Drosophila uncovers taste discrimination between different classes of fatty acids

Chemosensory systems are critical for evaluating the caloric value and potential toxicity of food prior to ingestion. While animals can discriminate between 1000s of odors, much less is known about the discriminative capabilities of taste systems. Fats and sugars represent calorically potent and innately attractive food sources that contribute to hedonic feeding. Despite the differences in nutritional value between fats and sugars, the ability of the taste system to discriminate between different rewarding tastants is thought to be limited. In Drosophila, sweet taste neurons expressing the Ionotropic Receptor 56d (IR56d) are required for reflexive behavioral responses to the medium-chain fatty acid, hexanoic acid. Further, we have found that flies can discriminate between a fatty acid and a sugar in aversive memory assays, establishing a foundation to investigate the capacity of the Drosophila gustatory system to differentiate between various appetitive tastants. Here, we tested whether flies can discriminate between different classes of fatty acids using an aversive memory assay. Our results indicate that flies are able to discriminate medium-chain fatty acids from both short- and long-chain fatty acids, but not from other medium-chain fatty acids. Characterization of hexanoic acid-sensitive Ionotropic receptor 56d (Ir56d) neurons reveals broad responsive to short-, medium-, and long-chain fatty acids, suggesting selectivity is unlikely to occur through activation of distinct sensory neuron populations. However, genetic deletion of IR56d selectively disrupts response to medium chain fatty acids, but not short and long chain fatty acids. These findings reveal Ir56d is selectively required for fatty acid taste, and discrimination of fatty acids occurs through differential receptor activation within shared populations of neurons. These findings uncover a capacity for the taste system to encode tastant identity within a taste category.

neuroscience↗